Download Mouse Hoxa2 mutations provide a model for microtia and auricle

4386 RESEARCH ARTICLE
Development 140, 4386-4397 (2013) doi:10.1242/dev.098046
© 2013. Published by The Company of Biologists Ltd
Mouse Hoxa2 mutations provide a model for microtia and
auricle duplication
Maryline Minoux1,2, Claudius F. Kratochwil1,3, Sébastien Ducret1, Shilu Amin4, Taro Kitazawa5,
Hiroki Kurihara5, Nicoletta Bobola4, Nathalie Vilain1 and Filippo M. Rijli1,3,*
SUMMARY
External ear abnormalities are frequent in newborns ranging from microtia to partial auricle duplication. Little is known about the
molecular mechanisms orchestrating external ear morphogenesis. In humans, HOXA2 partial loss of function induces a bilateral
microtia associated with an abnormal shape of the auricle. In mice, Hoxa2 inactivation at early gestational stages results in external
auditory canal (EAC) duplication and absence of the auricle, whereas its late inactivation results in a hypomorphic auricle, mimicking
the human HOXA2 mutant condition. By genetic fate mapping we found that the mouse auricle (or pinna) derives from the Hoxa2expressing neural crest-derived mesenchyme of the second pharyngeal arch, and not from a composite of first and second arch
mesenchyme as previously proposed based on morphological observation of human embryos. Moreover, the mouse EAC is entirely
lined by Hoxa2-negative first arch mesenchyme and does not develop at the first pharyngeal cleft, as previously assumed. Conditional
ectopic Hoxa2 expression in first arch neural crest is sufficient to induce a complete duplication of the pinna and a loss of the EAC,
suggesting transformation of the first arch neural crest-derived mesenchyme lining the EAC into an ectopic pinna. Hoxa2 partly
controls the morphogenesis of the pinna through the BMP signalling pathway and expression of Eya1, which in humans is involved
in branchio-oto-renal syndrome. Thus, Hoxa2 loss- and gain-of-function approaches in mice provide a suitable model to investigate
the molecular aetiology of microtia and auricle duplication.
INTRODUCTION
The external ear is composed of the auricle (or pinna) and the
external auditory canal (EAC). A variety of factors can affect its
morphogenesis, inducing a wide range of abnormalities such as
microtia and partial auricle duplications (Alasti and Van Camp,
2009; Baschek et al., 2006; Gore et al., 2006; Hunter and
Yotsuyanagi, 2005; Ku et al., 1998; Mishra and Misra, 1978; Pan et
al., 2010). Microtia, which is characterised by a small, abnormally
shaped auricle, is one of the most common external ear
abnormalities; the estimated prevalence ranges from 0.8 to 4.2 per
10,000 births depending on the population (Alasti and Van Camp,
2009). External ear abnormalities can occur as the only clinical
defect, but in most cases they appear in complex syndromes in
which others organs and structures are affected. External ear
anomalies are indeed commonly associated with internal and/or
middle ear dysplasia, resulting in hearing loss and problems in
speech and language development (Kountakis et al., 1995).
Deciphering the molecular mechanisms involved in external ear
morphogenesis is crucial not only to better understand the defaults
affecting the external ear, but also to gain a more comprehensive
view of the mechanisms involved in the genetic syndromes that
encompass external ear abnormalities.
1
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058
Basel, Switzerland. 2INSERM UMR 1121, Université de Strasbourg, Faculté de
Chirurgie Dentaire, 1, place de l’hôpital, 67 000 Strasbourg, France. 3University of
Basel, CH-4056 Basel, Switzerland. 4School of Dentistry, Faculty of Medical and
Human Sciences, University of Manchester, Manchester M13 9PT, UK. 5Department
of Physiological Chemistry and Metabolism, Graduate School of Medicine, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
*Author for correspondence ([email protected])
Accepted 19 August 2013
Genetic diagnostics in humans, as well as loss-of-function
experiments in mice, have begun to identify signalling factors and
transcriptional regulators involved in external ear morphogenesis.
Among these factors, Hoxa2 plays a crucial role. In humans, a
HOXA2 mutation induces a bilateral microtia associated with
abnormal shape of the auricle (Alasti et al., 2008). Moreover, hearing
impairment and partial cleft palate have been reported (Alasti et al.,
2008). A role for Hoxa2 in external ear morphogenesis is also
observed in mice, where its inactivation induces duplication of the
EAC and absence of the pinna (Gendron-Maguire et al., 1993; Mallo
and Gridley, 1996; Mark et al., 1995; Rijli et al., 1993). This
phenotype is associated with other craniofacial abnormalities, notably
a cleft palate and middle ear structure malformations. Indeed, Hoxa2
inactivation results in morphological transformation of the neural
crest-derived skeletal elements of the second pharyngeal arch,
including the middle ear ossicle stapes, into a duplicated set of first
arch-like elements, including duplication of the middle ear ossicles
malleus and incus (Gendron-Maguire et al., 1993; Rijli et al., 1993).
By temporally controlled inactivation, we have further shown that
pinna morphogenesis requires Hoxa2 function through advanced
developmental stages (Santagati et al., 2005). Whereas Hoxa2
inactivation before E11.5 results in the absence of the pinna, Hoxa2
inactivation at later stages (between E12.5 and E13.5) results in a
hypomorphic pinna (Santagati et al., 2005), thus mimicking the
human HOXA2 mutant condition. Altogether, these data emphasize
the major role of Hoxa2 in auricle morphogenesis. However, the
molecular programme that is regulated by Hoxa2 is only beginning to
be elucidated (Donaldson et al., 2012) and remains largely unknown.
Furthermore, it is unclear whether Hoxa2 is not only necessary but
also sufficient to induce and orchestrate the whole developmental
programme underlying pinna morphogenesis.
By genetic fate mapping, we show here that the mouse pinna
derives from the second pharyngeal arch Hoxa2-expressing neural
Development
KEY WORDS: Hox, BMP, Craniofacial development, External auditory canal, Neural crest, Pharyngeal arch, Pinna, Auricle, External ear
Hoxa2 and outer ear development
RESEARCH ARTICLE 4387
crest cells (NCCs), and not from a combined contribution of first
and second arch neural crest-derived mesenchyme, as proposed by
some authors based on morphological observations of human
embryos (reviewed by Hunter and Yotsuyanagi, 2005; Alasti and
Van Camp, 2009; Klockars and Rautio, 2009; Passos-Bueno et al.,
2009; Porter and Tan, 2005; Schoenwolf and Larsen, 2009). We
further show that the mouse EAC is entirely lined by Hoxa2negative first arch mesenchyme, and does not develop, as previously
proposed, at the first pharyngeal cleft (Jakubíková et al., 2005;
Schoenwolf and Larsen, 2009). By a conditional gain-of-function
approach, we show that ectopic Hoxa2 expression in first arch
NCCs is alone sufficient to induce the transformation of the neural
crest-derived first arch mesenchyme lining the EAC into a mirrorimage duplication of the pinna. Functional and molecular analyses
revealed that Hoxa2 controls the formation of the pinna through the
BMP signalling pathway by regulating the expression of bone
morphogenetic protein 5 (Bmp5), Bmp4 and twisted gastrulation
(Tsg; Twsg1 – Mouse Genome Informatics). Chromatin
immunoprecipitation and parallel sequencing (ChIP-Seq) on second
arch cells additionally shows that Hoxa2 binds to Bmp4 and Bmp5
non-coding regions, suggesting that they are direct targets. Bmp5
inactivation results in a small pinna [known as the short ear
mutation (King et al., 1994; Kingsley et al., 1992)], and we further
show that Bmp4 conditional inactivation also partially impairs pinna
development. Moreover, Hoxa2 regulates the expression of eyes
absent 1 (Eya1), which in humans is involved in the branchio-otorenal syndrome (Abdelhak et al., 1997; Kochhar et al., 2007). Thus,
Hoxa2 is a fundamental transcriptional regulator orchestrating the
morphogenesis of the auricle. This genetic approach in the mouse
might therefore represent a suitable model with which to understand
the aetiology of human auricle abnormalities.
in 4% PFA, rinsed in PBS at room temperature and incubated for 1 hour in
PBS at 65°C to inactivate endogenous alkaline phosphatase. Sections were
rinsed in a solution of NTMT [0.1 M NaCl, 0.1 M Tris-HCl (pH 9.5), 0.05
M MgCl2, 0.1% Tween 20]. For the staining, 3.5 μl NBT (Roche 1383213)
and 3.5 μl BCIP (Roche 1383221) were used per ml of NTMT. Sections
were rinsed in water, then in 100% ethanol, and mounted onto slides.
MATERIALS AND METHODS
In situ hybridisation
To fate map the external ear, the Z/AP (Lobe et al., 1999) and Rosa-CAGLSL-tdTomato (Ai14) (Madisen et al., 2010) reporter mouse lines were
crossed with the R4::Cre (Oury et al., 2006) mouse line. The Hoxa2EGFP and
Hoxa2EGFP(lox-neo-lox) knock-in mouse lines were described previously
(Pasqualetti et al., 2002). Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ embryos were
obtained by crossing Hoxa2EGFP(lox-neo-lox)/+ mice with Wnt1::Cre mice
(Danielian et al., 1998). The following lines were also used: CMV::CreERT2
(Santagati et al., 2005), Hoxa2lox (Ren et al., 2002), Bmp4loxP-lacZ [(Kulessa
and Hogan, 2002); hereafter referred to as Bmp4lox]. Hoxa2del/+ or Bmp4del/+
alleles were generated by Cre-mediated deletion, mating CMV::Cre (Dupé
et al., 1997) with Hoxa2lox/lox or Bmp4lox/lox mice, respectively. To generate
CMV::CreERT2;Hoxa2lox/del embryos, Hoxa2lox/lox or Hoxa2lox/+ were
crossed with CMV::CreERT2;Hoxa2del/+ mice. To generate Bmp4lox/del
embryos, Bmp4lox/lox were crossed with Bmp4del/+ mice. To generate
Wnt1::CreHoxa2-IRES-EGFP mice, the Rosa(lox-stop-lox)Hoxa2-IRES-EGFP/+ line
(Miguez et al., 2012) was crossed with the Wnt1::Cre line. All animal
experiments were approved by the Basel Cantonal Veterinary Authorities
and conducted in accordance with the Guide for Care and Use of Laboratory
Animals.
Tamoxifen treatment
Tamoxifen (TM) (Sigma) was dissolved at 20 mg/ml in pre-warmed corn oil
(Sigma) and stored at 4°C. Three successive TM administrations (10 mg at
E12.5, E13.0 and E13.5) were administered to pregnant females by oral
gavage with 12-hour intervals.
Alkaline phosphatase staining
E14.5 mouse fetuses were fixed overnight in 4% paraformaldehyde (PFA),
rinsed in PBS, equilibrated in 20% sucrose and embedded in Cryomatrix
(Thermo Electron Corporation). Cryostat sections (20 μm) were cut in the
frontal and horizontal planes (see Fig. 1A). Sections were fixed for 1 hour
Immunostaining for EGFP on cryosections was performed using a
polyclonal rabbit anti-EGFP antibody (Molecular Probes) and a peroxidaseconjugated goat anti-rabbit IgG (Beckman Coulter) or an Alexa 488conjugated secondary antibody (Invitrogen). For peroxidase-conjugated
goat anti-rabbit IgG, detection was performed with DAB chromogen
(DAKO). Immunostaining for RFP on cryosections was performed using a
polyclonal rabbit anti-RFP antibody (Rockland) and an Alexa 568conjugated secondary antibody (Invitrogen). Immunostaining for Ki67 was
performed on paraffin sections using a rabbit anti-Ki67 antibody
(Novocastra NCL-Ki67p) and a biotinylated anti-rabbit antibody. After
incubating for 30 minutes at room temperature with the VECTASTAIN
ABC reagent (Vector Labs), peroxidase activity was revealed with DAB
chromogen. For phosphorylated Smad (phosphoSmad) staining, cryostat
sections were incubated with phosphoSmad1/5/8 antibody (Cell Signaling)
at 37°C for 3 hours, followed by Alexa 488-conjugated secondary antibody
(Invitrogen).
Three-dimensional reconstruction of tissue sections
Consecutive cryostat sections (25 µm) of E14.5 Hoxa2EGFP/+ and
Hoxa2EGFP/EGFP fetuses, immunostained for EGFP, were imaged with a
Leica fluorescence macroscope. Between 49 and 68 sections were aligned
using Bitplane AutoAligner 6.0.1 (manual alignment). Structures were
artificially labelled in separated channels in Adobe Photoshop CS5.1. The
artificially labelled structures, as well as the GFP channel, were transformed
into surfaces in Bitplane Imaris 7.5.2 (surface area detail level: 35 µm;
thresholding: absolute intensity).
In situ hybridisation on frontal and horizontal sections (see Fig. 1A) were
performed as described (Santagati et al., 2005). The following RNA probes
were used: Hoxa2 (Ren et al., 2002), Eya1 (Xu et al., 1999), Bmp5
(Solloway and Robertson, 1999), Bmp4 (Hogan et al., 1994), Tsg (Zakin
and De Robertis, 2004), Prrx1, Prrx2 (ten Berge et al., 1998) and Tshz2
(Caubit et al., 2000).
ChIP analysis
ChIP experiments were performed on second pharyngeal arches isolated
from E11.5 CD1 embryos as described (Donaldson et al., 2012).
Immunoprecipitated DNA was subjected to qPCR using the following
primers: Bmp4, forward 5⬘-TGTGGGATAAAACAGGAGTGC-3⬘ and
reverse 5⬘-GCTCCCTCAGTTTGGCTAGA-3⬘; Bmp5, forward 5⬘TATGCAGTCTAGGGCCACCT-3⬘ and reverse 5⬘-CATTTGGGATAAAAGAACCTCAA-3⬘.
RESULTS
The whole mouse pinna derives from Hoxa2+
second arch NCCs
External ear morphogenesis occurs early in development, starting at
E12.0 in mouse or at the sixth week in humans. In humans, the
auricle was proposed to derive from tubercles, or hillocks, which
originate from spatially segregated NCC populations of both first
and second pharyngeal arches (reviewed by Hunter and
Yotsuyanagi, 2005). To map second arch neural crest contribution
to the mouse pinna, we crossed a rhombomere (R)4::Cre mouse line
(Oury et al., 2006) with the Z/AP (Lobe et al., 1999) or Rosa-CAGLSL-tdTomato (Ai14) (Madisen et al., 2010) reporter lines. Upon
Cre-mediated recombination, Alkaline phosphatase (AP) or
tdTomato gene expression is permanently activated in R4-derived
Development
Mouse lines and mating schemes
Immunohistochemistry
4388 RESEARCH ARTICLE
Development 140 (21)
NCC progenitors, thus allowing second arch NCC contribution to
the external ear to be assessed. Although the presence of a few
spared unstained cells cannot be ruled out, careful observation of
consecutive sections (Fig. 1D,E), as well as high magnifications of
co-stainings of tdTomato with DAPI to identify cell nuclei
(supplementary material Fig. S1), revealed that the whole mouse
pinna is contributed by R4-derived second arch NCCs, and not by
a composite of spatially segregated first and second arch NCCderived mesenchyme.
Hoxa2 is expressed in second, but not in first, arch NCCs
(reviewed by Minoux and Rijli, 2010). To map the contribution of
Hoxa2-expressing (Hoxa2+) NCCs to the pinna, we crossed the
Wnt1::Cre driver (Danielian et al., 1998), which expresses Cre in
NCC progenitors, to the Hoxa2EGFP(lox-neo-lox) allele (Pasqualetti et
al., 2002), in which EGFP is knocked in at the Hoxa2 locus and is
conditionally induced by Cre-mediated excision. In E14.5
Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ fetuses, the pinna is composed of
EGFP-expressing NCCs (Fig. 1F,I,J), indicating that this structure
mainly originates from Hoxa2+ NCC progenitors. Moreover, high
magnification of DAPI/EGFP co-stainings (supplementary material
Fig. S1) revealed that virtually all cells in the pinna are EGFP+, i.e.
they derive from Hoxa2+ NCC progenitors. Together with the R4
fate mapping, these results indicate that the mouse pinna mainly
originates from Hoxa2+ second arch NCCs.
The EAC develops from first arch Hoxa2–
ectomesenchyme
In humans, the EAC, an ectodermal structure lined by NCC-derived
mesenchyme, has been proposed to originate at the first pharyngeal
cleft, i.e. at the interface between the first and second arches, and to
Development
Fig. 1. Fate map of the mouse external ear. (A) Drawing representing a lateral view of the head of a newborn mouse. Blue and red lines indicate the
frontal and horizontal section planes used in this study. (B-E) Z/AP staining performed on frontal (B-D) and horizontal (E) sections through the external
ear of E14.5 R4::Cre;Z/AP fetuses. (F,K,P) Lateral views under the epifluorescence macroscope of the pinna of E14.5 Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ (F),
Hoxa2EGFP/+ (K) and Hoxa2EGFP/EGFP (P) fetuses. (G-J,L-O,Q-T) Anti-EGFP immunostaining on frontal (G-I,L-N,Q-S) and horizontal (J,O,T) sections through the
external ear of E12.5 Hoxa2EGFP/+ (O), E14.5 Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ (G-J), E14.5 Hoxa2EGFP/+ (L-N) and E14.5 Hoxa2EGFP/EGFP (Q-T) fetuses. In
B,C,G,H,J,L,M, note that the EAC is lined by EGFP– cells. In Q-T, note that the duplicated EAC (EAC*) is lined by EGFP+ cells. In O, the arrowhead maps the
position of vestigial first pharyngeal cleft at the interface between EGFP+ and EGFP– territories. In B and C, the otic capsule is outlined and arrowheads
indicate the membranous labyrinth. In horizontal sections, top is anterior, bottom is posterior. Frontal sections are from anterior to posterior, top is
dorsal, bottom is ventral. A, anterior; AT, auditory tube; D, dorsal; EAM, external auditory meatus; EAC, external auditory canal; H, hindbrain; P, posterior;
Pi, pinna; S, stapes; V, ventral.
be surrounded by NCCs contributed by both arches (Jakubíková et
al., 2005; Schoenwolf and Larsen, 2009). Contrary to this
prediction, we found that in E14.5 Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+
mouse fetuses the EAC and its surrounding mesenchyme are
entirely composed of Hoxa2–/EGFP– cells, and therefore not
derived from the second arch (Fig. 1G,H,J). Moreover, we observed
a sharp spatial segregation between Hoxa2–/EGFP– and
Hoxa2+/EGFP+ cell populations contributing to the EAC and the
pinna, respectively (Fig. 1J). A similar cell sorting was observed in
E14.5 Hoxa2EGFP/+ fetuses in which EGFP is knocked in at the
Hoxa2 locus and faithfully recapitulates the Hoxa2 expression
domain (Pasqualetti et al., 2002) (Fig. 1K-N). Analysis at E12.5
confirmed that mouse EAC begins to form entirely anterior to the
second arch-derived Hoxa2+/EGFP+ cells that form the pinna,
within the Hoxa2–/EGFP– first arch-derived tissue (Fig. 1O). Thus,
at E12.5 the EAC and the pinna develop opposite to and almost
equidistant from the vestigial first pharyngeal cleft that maps at the
border between EGFP+ and EGFP– territories (arrowhead,
Fig. 1O); notably, their respective cell contributions from the first
and second arches never intermingle despite the complex
morphogenetic cell movements that occur during their formation.
To investigate the spatial distribution of second arch cells in the
absence of Hoxa2 function, we used Hoxa2EGFP/EGFP homozygous
mutant embryos (Fig. 1P). By 3D reconstruction of tissue sections,
we confirmed the previously described duplication of the EAC
(Gendron-Maguire et al., 1993; Mallo and Gridley, 1996; Mark et
al., 1995; Rijli et al., 1993) (EAC*, Fig. 2) and found that the EAC*
Fig. 2. Three-dimensional reconstruction of the external ear of
Hoxa2EGFP/+ and Hoxa2EGFP/EGFP fetuses. (A-C) 3D reconstruction of the
external auditory canal (EAC; red), tympanic bone (TB; yellow) and EGFP+
domain (green) in E14.5 Hoxa2EGFP/+ control heterozygous fetuses.
(D-F) 3D reconstruction of the EAC and its duplicated counterpart (EAC*;
red), the tympanic bone and its duplicated counterpart (TB*; yellow) and
EGFP+ domain (green) in E14.5 Hoxa2EGFP/EGFP homozygous mutant
fetuses.
RESEARCH ARTICLE 4389
develops within the Hoxa2+/EGFP+ domain (Fig. 2, see also
Fig. 1Q-T). By contrast, the orthotopic EAC fully develops into the
Hoxa2–/EGFP– territory, both in E14.5 Hoxa2EGFP/+ control and
Hoxa2EGFP/EGFP homozygous mutant fetuses (Fig. 2, see also
Fig. 1L,M,T). The border between EGFP+ and EGFP– territories
appears to be the axis of symmetry, on either side of which the EAC
and its ectopic EAC* counterpart develop (Fig. 1T). Tissue sections
additionally show that in E14.5 Hoxa2EGFP/EGFP homozygous
mutant fetuses, the EGFP+ cells maintain their normal spatial
segregation and do not ectopically intermingle with EGFP– cells,
despite the fact they have acquired a first arch-like identity (Fig. 1T).
Hoxa2 organises spatial patterns of cell
proliferation during external ear morphogenesis
At E14.5, Hoxa2 expression extensively overlaps with the
developing pinna. On horizontal and frontal sections, the Hoxa2+
domain contains an outer territory, which includes the pinna, and
an inner territory (Fig. 3B,H, the dashed line delimits the territories).
The inner domain is strongly Hoxa2+, whereas the outer domain
displays sparser Hoxa2 transcript distribution (Fig. 3B,H). The two
Hoxa2+ territories roughly abut at the base of the bending pinna
(arrows, Fig. 3B,H). Hoxa2 is additionally expressed in the
mesenchyme at the tip and below the ectoderm on both the dorsal
and ventral sides of the pinna (Fig. 3B,H).
The spatial distribution of Hoxa2 transcripts suggests distinct
proliferation/differentiation states of cellular subsets. In E14.5
control fetuses, Ki67+ proliferating cells are present at the tip and are
orderly aligned on both the dorsal and ventral sides of the pinna,
below the ectoderm, whereas the mesenchymal core of the pinna is
only contributed by Ki67– postmitotic differentiating cells
(Fig. 3A,C,G). At the pinna distal edge (tip), the Ki67+ cell pattern
overlaps with the Hoxa2 transcript distribution (Fig. 3A,B),
indicating that Hoxa2 is mainly expressed in proliferating NCCs.
By contrast, the inner Hoxa2 expression domain encompasses both
Ki67+ and Ki67– cell subsets (Fig. 3A,B,G,H), with Ki67+ cells
orderly aligned (arrows, Fig. 3A,C,G) and adjacent to Ki67–
differentiating mesenchymal cells. In addition, at E14.5 a subset of
spatially organised Ki67+ proliferating cells at the base and all along
the dorsal aspect of the pinna (arrowheads, Fig. 3A,C,G) does not
express Hoxa2 (Fig. 3A,B,G,H). This latter population of highly
proliferating cells selectively expresses Eya1 (Fig. 3G,I), which is
essential for pinna development in mice (Xu et al., 1999).
Interestingly, at the beginning of pinna morphogenesis (E12.0-12.5),
Eya1 and Hoxa2 appear to be co-expressed in a subset of second
arch-derived NCCs at the base of the future pinna (Fig. 3M-O),
which are likely to correspond to the precursors of the Eya1+
population observed at later stages (Fig. 3I). In E12.5 Hoxa2
homozygous mutant embryos, this early Eya1 expression domain
is lacking (Fig. 3P,Q).
In control Hoxa2lox/del fetuses, which bear both a fully deleted
and a floxed Hoxa2 allele (Ren et al., 2002), the pinna is of normal
appearance (Santagati et al., 2005) (Fig. 3A-C), indicating that
Hoxa2 haploinsufficiency in mouse does not result in gross external
ear defects. By contrast, tamoxifen (TM) administration to
CMV::CreERT2;Hoxa2lox/del fetuses at NCC postmigratory stages
(E12.5, E13.0 and E13.5) induces a hypomorphic pinna (Santagati
et al., 2005) (supplementary material Fig. S2), thus bypassing an
early role of Hoxa2 and supporting a late requirement in pinna
morphogenesis. The smaller and dysmorphic pinna of TM-treated
CMV::CreERT2;Hoxa2lox/del mutant newborns (supplementary
material Fig. S2) is reminiscent of the HOXA2 mutant phenotype
in humans (Alasti et al., 2008). Analysis by in situ hybridisation
Development
Hoxa2 and outer ear development
4390 RESEARCH ARTICLE
Development 140 (21)
Fig. 3. Hoxa2 organises spatial patterns of cell proliferation in the
pinna. (A-L) Anti-Ki67 immunostaining (A,C-G,J,K) and Hoxa2 (B,H) and
Eya1 (I,L) in situ hybridisation on frontal (A-F) and horizontal (G-L) sections
through the external ear of E14.5 Hoxa2lox/del control (A-C), wild-type (WT)
(G-I) and CMV::CreERT2;Hoxa2lox/del (D-F,J-L) tamoxifen (TM)-treated fetuses
at E12.5, E13.0 and E13.5. Frontal sections are from anterior to posterior,
top is dorsal, bottom is ventral. In horizontal sections, top is anterior,
bottom is posterior. In B and H, the dashed line separates outer from
inner Hoxa2+ territories, abutting at the base of the bending pinna
(arrows). In A,C,G, arrows indicate orderly aligned proliferating cells in the
inner Hoxa2+ domain; arrowheads indicate orderly aligned proliferating
cells at the base of the pinna and extending along its dorsal aspect. In DF,J, arrows and arrowheads indicate altered spatial segregation of Ki67+
and Ki67– mesenchymal cells in the pinna (E,F) and in the inner domain
(D-F,J), respectively. Asterisks indicate an unpatterned mass of
proliferating cells accumulating at the dorsal base of the pinna. (M-O) In
situ hybridisation on adjacent horizontal sections through the external
ear of E12.5 wild-type embryo using Hoxa2 (M) and Eya1 (N) probes. In O,
the images in M and N have been merged to highlight Hoxa2 and Eya1
co-expression in a subset of second arch NCCs (arrow). Top is anterior,
bottom is posterior. (P,Q) Eya1 whole-mount in situ hybridisation on E12.5
wild-type (P) and Hoxa2 homozygous mutant (Q) embryos. OC, otic
capsule; Pi, pinna.
confirmed the almost complete loss of Hoxa2 expression in TMtreated mutants when compared with control embryos
(supplementary material Fig. S3).
At E14.5, no apoptotic cells were observed in control or in TMtreated CMV::CreERT2;Hoxa2lox/del mutant fetuses (not shown),
Hoxa2 regulates Bmp5 and Bmp4 expression in
the developing pinna
The hypomorphic pinna in TM-treated CMV::CreERT2;Hoxa2lox/del
fetuses is reminiscent of the phenotype of the short ear mutation,
which inactivates Bmp5 (King et al., 1994; Kingsley et al., 1992).
Thus, Bmp5 is a suitable candidate to be regulated by Hoxa2. In E14.5
control pinna, Bmp5 expression is restricted to the mesenchymal cores
of the inner and outer Hoxa2+ domains, although not at the tip or in
most of the proliferating mesenchyme of the pinna (Fig. 4A,B,E,F).
In TM-treated CMV::CreERT2;Hoxa2lox/del fetuses, Bmp5 expression
levels were strongly reduced in the pinna (arrowheads, Fig. 4M,N).
We also observed a reduction in the spatial extent of Bmp5 expression
in the inner Hoxa2+ domain (arrows, Fig. 4M,N), with an
accumulation of Bmp5 residual expression at the base of the pinna
(asterisk, Fig. 4M). Hoxa2 temporal inactivation selectively
downregulates Bmp5, although not paired related homeobox 1
(Prrx1), which encodes a transcription factor involved in craniofacial
development (Martin et al., 1995) (Fig. 4I,J,Q,R).
Development
suggesting that the smaller dysmorphic pinna is not the result of
increased cell death. This was also indirectly supported by the
persistence of EGFP+ cells in the outer ear region of
Hoxa2EGFP/EGFP full knockout fetuses, which lack a normal pinna
(Fig. 1P-T). The hypomorphic pinna of E14.5 TM-treated CMVCreERT2;Hoxa2lox/del fetuses lacks the normal spatial segregation
between Ki67+ and Ki67– mesenchymal cells (arrows, Fig. 3E,F).
Moreover, a mass of proliferating cells abnormally accumulates at
the dorsal base of the pinna (asterisks, Fig. 3D-F,J), and the inner
Hoxa2+ domain appears disorganised, with fewer Ki67+ cells and an
accumulation of unpatterned Ki67– differentiating cells
(arrowheads, Fig. 3D-F,J). Lastly, in E14.5 TM-treated CMVCreERT2;Hoxa2lox/del fetuses, the population of spatially organised
proliferating cells that expresses Eya1 is still present (Fig. 3L),
suggesting a role of Hoxa2 in establishing, but not maintaining, the
proliferative Eya1+ territory.
Altogether, these results indicate that, after E12.5, Hoxa2
contributes to the normal size and shape of the pinna by organising
spatially restricted local patterns of cell proliferation.
Hoxa2 and outer ear development
RESEARCH ARTICLE 4391
Fig. 4. Hoxa2 positively regulates Bmp5 and Bmp4 expression.
(A-T) In situ hybridisation on horizontal (A,C,E,G,I,K,M,O,Q,S) and frontal
(B,D,F,H,J,L,N,P,R,T) sections through the external ear of E14.5 control (A-L)
and CMV::CreERT2;Hoxa2lox/del mutant fetuses treated with tamoxifen (TM)
at E12.5, E13.0 and E13.5 (M-T), using Hoxa2 (A-D), Bmp5 (E,F,M,N), Bmp4
(G,H,O,P), Prrx1 (I,J,Q,R), Tshz2 (K,S) and Prrx2 (L,T) probes. In M-P,
arrowheads indicate the reduction of Bmp5 and Bmp4 expression levels in
the pinna, arrows indicate the reduction in the spatial extent of Bmp5
(M,N) and Bmp4 (O,P) expression in the inner Hoxa2+ domain, and the
asterisk indicates the accumulation of Bmp5 residual expression at the
base of the pinna (M) in TM-treated mutants. Note that the section in D is
the same as that in Fig. 3D, and is adjacent to H. In frontal sections, top is
dorsal and bottom is ventral. In horizontal sections, top is anterior and
bottom is posterior. OC, otic capsule; Pi, pinna.
We next analysed Bmp4 expression. At E12.0-12.5, Bmp4 is
expressed at the distal edge (tip) of the forming pinna, as well as in
a small population of mesenchymal cells at its base (not shown).
This pattern is maintained and extended at E13.5 and E14.5,
becoming similar to the Hoxa2 expression pattern. Specifically,
Bmp4 is highly expressed in the mesenchymal core of the inner
Hoxa2+ domain (Fig. 4C,D,G,H). In the pinna, Bmp4 is expressed
at the tip and in the proliferating mesenchyme below the dorsal and
Temporal inactivation of Hoxa2 results in altered
BMP signalling in the developing pinna
The activity of BMPs is modulated in the extracellular space through
their interaction with secreted agonists or antagonists. Tsg is a secreted
BMP modulator involved in mouse head development (Zakin and De
Robertis, 2004). Although no external ear defects are observed in
Tsg−/− or Bmp4+/− mice, compound Tsg−/−;Bmp4+/− newborns
display, in severe cases, low-set ears associated with an abnormal
shape of the pinna (Zakin and De Robertis, 2004), indicating that Tsg
is involved in modulating Bmp4 activity during pinna development.
In E14.5 wild-type fetuses, Tsg is strongly expressed in a stripe of
Ki67+ highly proliferating cells in the inner Hoxa2+ domain
(supplementary material Fig. S5K, arrow; Fig. 3G, arrow), directly
adjacent to the Bmp4+ mesenchymal core (supplementary material
Fig. S5A-O). In E14.5 TM-treated CMV::CreERT2;Hoxa2lox/del pinna,
Tsg is severely downregulated (supplementary material Fig. S5P-R).
These results, together with the downregulation of Bmp4 and Bmp5
expression and the effects on cell proliferation patterns (Figs 3, 4),
further support the notion that Hoxa2 is involved in BMP signalling
regulation during pinna formation.
To further investigate the downstream effects induced by Bmp5,
Bmp4 and Tsg downregulation in Hoxa2 mutants, we analysed the
phosphorylation of Smad1, 5 and 8, a hallmark of canonical BMP
signal transduction (Heldin and Moustakas, 2012; Horbelt et al.,
2012), by anti-phosphoSmad1/5/8 immunostaining. In E14.5
control fetuses, the phosphoSmad1/5/8+ cell distribution is spatially
restricted. PhosphoSmad1/5/8+ cells are observed throughout the
Bmp5+/Bmp4+ territory of the inner Hoxa2+ domain extending to
the base of the pinna (white arrows, Fig. 5A,C; supplementary
material Fig. S4C), although not in the Bmp5+/Bmp4– mesenchymal
core of the developing pinna (Fig. 5A,C; supplementary material
Fig. S4A-C). PhosphoSmad1/5/8+ cells are also detected in the
Ki67+/Bmp4+ proliferating domain at the tip of the pinna (green
arrowheads, Fig. 5A,C; supplementary material Fig. S4A,C), and
in the Eya1+/Ki67+ highly proliferative domain abutting the pinna
mesenchymal core (white arrowheads, Fig. 5A,C; supplementary
material Fig. S4C).
In E14.5 TM-treated CMV::CreERT2;Hoxa2lox/del pinna, costaining between phosphoSmad1/5/8 antibody and DAPI shows that
phosphoSmad1/5/8+ domains at the base and at the tip of the pinna
are either strongly reduced or absent (white arrows and green
arrowheads, Fig. 5E-H). These data indicate that Hoxa2 temporal
inactivation has strong effects on the local regulation of canonical
BMP signalling. By contrast, the Eya1+ subpopulation of
phosphoSmad1/5/8+ cells is still present (white arrowheads,
Fig. 5E,G), in keeping with the observation that the late Hoxa2
inactivation only partially affects this Eya1+ cell population (see
above; Fig. 3I,L).
Development
ventral ectoderm (Fig. 4G,H). This transcript distribution is
complementary to that of Bmp5 (supplementary material
Fig. S4A,B); notably, the Bmp4 and Bmp5 expression patterns
together recapitulate the Hoxa2 expression pattern in the pinna
(Fig. 4A-H). In E14.5 TM-treated CMV::CreERT2;Hoxa2lox/del
mutant pinna, Bmp4 expression is absent or strongly reduced
(Fig. 4O,P), whereas Prrx1 (Fig. 4J), Prrx2 (Fig. 4L) (ten Berge et
al., 1998) and teashirt zinc finger family member 2 (Tshz2) (Caubit
et al., 2000) (Fig. 4K) are still expressed in the inner Hoxa2+ domain
and at the tip of the pinna (Fig. 4S,T; data not shown).
In summary, the above results indicate that Hoxa2 is required to
maintain the normal levels and spatial distribution of Bmp4 and
Bmp5 transcripts during pinna growth and morphogenesis.
4392 RESEARCH ARTICLE
Development 140 (21)
Fig. 5. Hoxa2 temporal inactivation affects
the BMP signalling pathway. (A-H) AntiphosphoSmad1/5/8 immunostaining (A,C,E,G)
and DAPI staining (B,D,F,H) of horizontal
sections through the external ear of E14.5
CMV::CreERT2 control (A-D) and
CMV::CreERT2;Hoxa2lox/del mutant (E-H) fetuses
treated with tamoxifen (TM) at E12.5, E13.0 and
E13.5. A and B, C and D, E and F, G and H are
the same sections co-stained for both antiphosphoSmad1/5/8 and DAPI. In E,G,
phosphoSmad1/5/8 staining is absent or
strongly reduced at the tip of the pinna (green
arrowheads) and in the inner Hoxa2+ domain
(white arrows), but is maintained in the Eya1+
subpopulation abutting the pinna
mesenchymal core (white arrowheads). Top is
anterior, bottom is posterior.
occurs at a low frequency (n=9/22) in these hypomorphic mutants.
Bilateral microphthalmia, smaller body size and subcutaneous
edema are also observed in most of the Bmp4lox/del hypomorphic
mutants (Fig. 6A,B). The weak penetrance of the pinna phenotype
is likely to be due to partial functional redundancy with other
BMP family members, such as Bmp5, and/or the persistence of
residual Bmp4 function in Bmp4lox/del hypomorphic mutants.
Bmp4 homozygous null mutants die around gastrulation (Winnier
et al., 1995), preventing analysis of its role in external ear
morphogenesis. Nonetheless, these results highlight Bmp4
involvement, at least to some extent, in the morphogenesis of the
pinna.
Fig. 6. Bmp4 involvement in pinna
morphogenesis. Lateral views of E16.5 (A,C,D)
Bmp4lox/+ control and (B,E,F) Bmp4lox/del
hypomorphic mutant fetuses. (C,D) Enlarged
views of the hindlimb and pinna of the fetus in
A. In B, the arrowhead indicates the apparent
lack of normal eyes, and the arrow points to a
small pinna, which is magnified in F. In E, the
arrows indicate polydactyly.
Development
Role of Bmp4 in external ear morphogenesis
Bmp4lox/+ (Kulessa and Hogan, 2002) heterozygous mutant mice
survive and do not show external ear defects (not shown). To
investigate the consequences of further reducing Bmp4 activity
during pinna morphogenesis, we generated Bmp4lox/del
hypomorphic mutants bearing both a fully deleted and a floxed
Bmp4 allele. Unlike null mutants, Bmp4lox/del fetuses survive until
E16.5, a stage at which the pinna has normally already folded over
the meatus (Fig. 6A,D). In severe cases, Bmp4lox/del fetuses display
a hypomorphic pinna (Fig. 6B,F), albeit at low frequency
(n=5/22). When present, this phenotype is always associated with
polydactyly (arrows, Fig. 6E, compare with 6C), which also
Hoxa2 binds to Bmp4 and Bmp5 non-coding
regions in second pharyngeal arch
By mining datasets from a genome-wide map of Hoxa2 binding to
chromatin (ChIP-Seq) from second pharyngeal arch tissue dissected
just prior to external ear formation (Donaldson et al., 2012), we
found binding of Hoxa2 in proximity to Bmp4 [72 kb downstream
of the Bmp4 transcription start site (TSS)] and within the third intron
of Bmp5 (52 kb upstream of the Bmp5 TSS). We confirmed Hoxa2
binding enrichment on both Bmp4 and Bmp5 regions by performing
conventional ChIP on second arches dissected from E11.5 wild-type
embryos (supplementary material Fig. S6A). The region bound by
Hoxa2 in the third intron of Bmp5 is the result of an insertion that
is present only in mice and rat. By contrast, the region located
downstream of the Bmp4 TSS is widely conserved in vertebrates
and contains putative Pbx/Hox and Hox binding sites
(supplementary material Fig. S6B). Moreover, in E11.5 Hoxa2
mutant second arch the expression of both Bmp4 and Bmp5 is
downregulated (Donaldson et al., 2012). Together with the
expression pattern changes observed in temporally induced Hoxa2
mutants (Fig. 4), these results suggest that Hoxa2 might directly
maintain Bmp4 and Bmp5 expression in spatially restricted domains
of the developing external ear.
Hoxa2 is sufficient to induce an ectopic pinna
To address whether Hoxa2 is not only necessary but also sufficient
for the formation of the pinna we established a conditional
overexpression system in the mouse allowing ectopic Hoxa2
expression in Hox-negative NCCs anterior to the second arch.
Conditional Hoxa2 overexpression in NCCs was induced by
mating the Wnt1::Cre line (Danielian et al., 1998) with a
Rosa(lox-stop-lox)Hoxa2-IRES-EGFP allele (Miguez et al., 2012) to produce
Wnt1::Cre;Rosa(lox-stop-lox)Hoxa2-IRES-EGFP (hereafter designated
Wnt1Hoxa2-IRES-EGFP).
Wnt1Hoxa2-IRES-EGFP fetuses die at birth and display craniofacial
defects, including face, middle ear and skull bone malformations, as
well as a reduction in lower jaw size (Fig. 7; data not shown). Most
notably, all of them (n=25/25) display an ectopic, fully formed pinna,
RESEARCH ARTICLE 4393
which is a mirror-image duplication of its normal orthotopic
counterpart (compare Fig. 7A with supplementary material Fig. S7BE). The ectopic pinna replaces the EAC, and thus is likely to form
from first arch NCCs. The orthotopic pinna, which is derived from the
second pharyngeal arch, appears normally shaped, indicating that
Hoxa2 overexpression from the Rosa(lox-stop-lox)Hoxa2-IRES-EGFP allele
within its own expression domain does not result in overt pinna
morphological abnormalities (Fig. 7B-E). It is also noteworthy that
Wnt1Hoxa2-IRES-EGFP fetuses occasionally display multiple additional
ectopic structures around the eye that morphologically resemble small
ectopic pinnae (arrows, Fig. 7C,D).
The presence of an internal ribosome entry site (IRES) in the
Rosa(lox-stop-lox)Hoxa2-IRES-EGFP allele allows the cells that are ectopically
expressing Hoxa2 to be traced by immunohistochemistry with an
anti-EGFP antibody. We therefore compared the EGFP+ cell
distribution in E14.5 Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ fetuses, in
which EGFP is conditionally expressed in NCCs expressing the
endogenous Hoxa2 (Fig. 8A), with Wnt1Hoxa2-IRES-EGFP fetuses, which
additionally ectopically express Hoxa2-IRES-EGFP in rostral Hoxa2negative territories. In Wnt1Hoxa2-IRES-EGFP mutants, the ectopic pinna
is entirely contributed by Hoxa2-IRES-EGFP-expressing cells
(Fig. 8B-D).
Moreover, the ectopic pinna of Wnt1Hoxa2-IRES-EGFP fetuses
displays mirror-image duplications of the Bmp5, Bmp4 and Tsg
expression patterns (Fig. 8E-L; supplementary material Fig. S7).
Namely, Bmp4 is expressed at the tip of the duplicated pinna as well
as in the mesenchyme at its base (Fig. 8I-L). Bmp5 and Tsg are
expressed in the mesenchymal core and at the base of the duplicated
pinna (Fig. 8E-H; supplementary material Fig. S7). Notably, Bmp4
is also expressed in the ectopic structures that form around the eye
(Fig. 8L), while Bmp5 is expressed in the underlying mesenchyme
(Fig. 8F-H). Strikingly, proliferating Ki67+ and differentiating
DAPI+ cell patterns are spatially organised in the ectopic pinna as a
faithful mirror image of the cellular organisation of the orthotopic
pinna (Fig. 8M-Q).
These results not only confirm the molecular identity of the
duplicated pinna but also demonstrate that Hoxa2 is sufficient to
Fig. 7. Hoxa2 expression is sufficient to induce
an ectopic pinna. (A-E) Lateral views of the head
of E18.5 (A,B,E) and E17.5 (C,D) wild-type (A) and
Wnt1Hoxa2-IRES-EGFP mutant (B-E) fetuses. The arrow
in B indicates a duplicated pinna, which is
enlarged in E. Arrows in C and D indicate ectopic
structures forming all around the eye that
resemble small ectopic pinnae.
Development
Hoxa2 and outer ear development
4394 RESEARCH ARTICLE
Development 140 (21)
induce and maintain the genetic programme that underlies the
formation of the pinna.
DISCUSSION
Revisiting the embryological origin of the
external ear
The human auricle cartilage has classically been proposed to derive
from six nodular masses of mesenchyme, termed the hillocks of His,
that appear during the sixth week of development, three in the first
(mandibular) and three in the second (hyoid) pharyngeal arch,
respectively (reviewed by Hunter and Yotsuyanagi, 2005;
Schoenwolf and Larsen, 2009). On the other hand, the ectodermal
EAC has been proposed to originate at the first pharyngeal cleft and
to be lined by NCCs contributed by both first and second arches
(Jakubíková et al., 2005; Schoenwolf and Larsen, 2009). The
hillocks eventually fuse to form the different parts of the human
external ear. Although there is consensus that the tragus, projecting
in front of the EAC, derives from mandibular hillocks, the
respective contributions of mandibular or hyoid hillocks to the
auricle are less clear (Hunter and Yotsuyanagi, 2005; Kagurasho et
al., 2012). For instance, according to some authors, but not others,
first arch-derived hillocks not only form the tragus but also give rise
to the helix, which is the auricle folded edge, and the antihelix
(reviewed by Hunter and Yotsuyanagi, 2005).
Our genetic fate mapping in the mouse provides novel insights into
the embryological origin of the external ear. We show that the
mesenchyme just anterior to the EAC (possibly, the mouse
homologue of the human tragus) is contributed by Hoxa2–/EGFP–
cells (Fig. 1), indicating that, as proposed in humans, it might
originate from first pharyngeal arch NCCs. By contrast, genetic fate
mapping and Hoxa2 functional analysis reveal for the first time that
the mouse pinna is entirely contributed by second arch Hoxa2+ neural
crest-derived mesenchyme, and not by both first and second arch
NCCs. Moreover, genetic fate mapping shows that the ectodermderived EAC invaginates into, and is entirely surrounded by, Hoxa2negative mesenchyme, which is both anterior and spatially segregated
from second arch-derived mesenchyme (Fig. 1), and is thus most
likely of first arch origin. Therefore, the mouse EAC appears to be a
first arch-derived structure that does not originate at the border
between first arch Hoxa2-negative and second arch Hoxa2-positive
mesenchyme, i.e. at the first pharyngeal cleft, as previously assumed
(Jakubíková et al., 2005; Schoenwolf and Larsen, 2009).
Development
Fig. 8. Molecular identity of Hoxa2-induced
pinna. (A-D) Anti-EGFP immunostaining on
horizontal sections through the external ear of
E14.5 Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ control (A)
and Wnt1Hoxa2-IRES-EGFP mutant (B-D) fetuses.
(E-L) Bmp5 (E-H) and Bmp4 (I-L) in situ
hybridisation on horizontal sections through
the external ear of E14.5 wild-type (E,I) and
Wnt1Hoxa2-IRES-EGFP mutant (F-H,J-L) fetuses. Arrows
indicate Bmp5 and Bmp4 ectopic expression in
the duplicated pinna (Pi*) and more anteriorly.
Asterisks indicate the ectopic structures that form
around the eye, one of which is enlarged in the
inset in L. Bmp4 is expressed in the ectopic
structures, whereas Bmp5 is expressed in the
mesenchyme just beneath. (M-Q) Anti-Ki67
immunostaining (M,N,P) and DAPI staining (O,Q)
on horizontal sections through the external ear of
E14.5 wild-type (M) and Wnt1Hoxa2-IRES-EGFP mutant
(N-Q) fetuses. Top is anterior, bottom is posterior.
Pi, pinna.
Auricle morphology is complex in humans, suggesting that
additional components might have been recruited from the
developing first arch mesenchyme, as compared with mouse.
Nonetheless, it is possible that, as in mouse, the human auricle is
mostly contributed by second arch NCCs. This conclusion may be
indirectly supported by the following arguments. First, there is
agreement among clinicians that the trigeminal (first arch) nerve
supply is limited to the tragus and anterior part of the EAC
(reviewed by Hunter and Yotsuyanagi, 2005; Wood-Jones and IChuan, 1934). Second, the auricle is not affected in otocephaly,
which is a genetic syndrome resulting from failure of first arch
development. Interestingly, only the tragus, which is known to
derive from the first arch, is absent in this syndrome (Hunter and
Yotsuyanagi, 2005; Wood-Jones and I-Chuan, 1934). Lastly, based
on the analysis of the recurrent localisation in the neighbourhood of
the tragus of frequent human congenital abnormalities such as preauricular fistulae and appendages, Wood-Jones and I-Chuan (WoodJones and I-Chuan, 1934) already came to the conclusion that the
human pinna is mainly of hyoid origin. This latter conclusion is now
fully supported by our current fate mapping in the mouse,
supporting evolutionary conservation of the pharyngeal arch
contribution to the definitive external ear in mammals. Overall, our
results could be of interest from a clinical standpoint because they
might facilitate our understanding of human syndromes, notably
concerning the correlation between the embryological origin of the
external ear, gene expression patterns, and the interpretation of the
phenotypic outcome of their disruption.
Revisiting the EAC phenotype of the Hoxa2
mutant mouse
By reconsidering the embryological origin of the EAC, our fate map
as well as 3D tissue reconstruction allow a better understanding of the
previously described mouse Hoxa2 knockout phenotype (Rijli et al.,
1993). The finding that the mouse EAC and its surrounding
mesenchyme derive from the first pharyngeal arch and do not develop
at the first pharyngeal cleft better explains the observed partial
duplication of the EAC in Hoxa2 mutants (Rijli et al., 1993). Our
analysis indicates that, rather than forming the EAC, the first
pharyngeal cleft maps at the border between EGFP+ and EGFP–
NCCs and could represent the axis of symmetry on either side of which
a mirror-image duplication of the EAC occurs in Hoxa2 mutant mice.
Hoxa2 is a major determinant of pinna formation
Hoxa2 inactivation in the mouse results in a mirror-image homeotic
transformation of the second arch-derived stapes, styloid process of
the temporal bone and lesser horn of the hyoid bone into a duplicated
set of first arch-like structures, namely the proximal part of the jaw
(Meckel’s) cartilage, the incus, malleus, tympanic bone and proximal
gonial bone (Gendron-Maguire et al., 1993; Rijli et al., 1993). These
structures are normally derived from the Hox-negative NCCs arising
from the rostral hindbrain (Köntges and Lumsden, 1996; Kuratani,
2005; Minoux and Rijli, 2010; Rijli et al., 1993; Santagati and Rijli,
2003; Takechi and Kuratani, 2010). This subset of first arch NCCs
and second arch NCCs share a common Hox-free ground (default)
patterning molecular programme upon which Hoxa2 expression
selects second arch identity (Köntges and Lumsden, 1996; Kuratani,
2005; Minoux et al., 2009; Minoux and Rijli, 2010; Rijli et al., 1993;
Santagati and Rijli, 2003; Takechi and Kuratani, 2010). However, the
possible extension of this model to other NCC-derived structures,
such as the external ear, was not previously assessed.
In Wnt1Hoxa2-IRES-EGFP embryos, ectopic Hoxa2 expression in
Hox-negative cranial NCCs induces abnormalities in structures
RESEARCH ARTICLE 4395
derived from midbrain and forebrain Hox-negative NCCs, such as
most of the jaw, facial and skull bones, supporting the notion that
ectopic Hox gene expression interferes with normal craniofacial
development (e.g. Couly et al., 1998). However, we found that
conditional ectopic Hoxa2 expression is also sufficient to repattern
mandibular arch mesenchyme, derived from rostral hindbrain
NCCs, and to generate a mirror-image transformation into a second
arch-like structure, the pinna, at the expense of the EAC. This
further extends the proposal of a Hox-free ground patterning
programme shared by hindbrain NCCs, and suggests that the first
arch-derived mesenchyme normally lining the EAC has been
homeotically transformed into a duplicated pinna, normally a
second arch derivative. Lastly, the ability of Hoxa2 to repattern (a
subset of) first arch NCCs appears to be conserved
(Grammatopoulos et al., 2000; Pasqualetti et al., 2000). In this
respect, we additionally observed in Wnt1Hoxa2-IRES-EGFP newborns
skeletal morphological changes that could be interpreted as partial
transformations of middle ear first arch skeletal elements into
second arch-like structures (not shown).
Several cases in the literature have reported partial or total
duplication of the human pinna (Baschek et al., 2006; Gore et al.,
2006; Hunter and Yotsuyanagi, 2005; Ku et al., 1998; Mishra and
Misra, 1978; Pan et al., 2010). Although the genetic basis of such a
phenotype has not been investigated, it is tempting to speculate that
ectopic HOXA2 expression might underlie the ‘polyotia’ or ‘mirror
ear’ phenotype observed in humans. As Hoxa2 alone is able to
induce the whole developmental programme underlying the
morphogenesis of the pinna, our data moreover suggest that
numerous genes involved in human auricle abnormalities are
HOXA2 targets.
Towards an understanding of the molecular
mechanisms involved in pinna morphogenesis
Our study provides novel insights into the largely unknown molecular
programme involved in external ear morphogenesis. The
identification of such a programme could improve our understanding
of the HOXA2 mutant phenotype in humans. Indeed, we have shown
that Hoxa2 regulates the expression of Eya1, which is involved in the
branchio-oto-renal syndrome in humans (Abdelhak et al., 1997;
Kochhar et al., 2007). Our functional and molecular analyses also
reveal that Hoxa2 is involved in the regulation of Bmp5, Bmp4 and
Tsg expression and Smad1/5/8 activity. Thus, Hoxa2 acts upstream of
the BMP canonical signalling pathway during external ear
morphogenesis. A recent study has reported a role for Hoxa2 in
activating the Wnt-β-catenin signalling pathway in the second arch
(Donaldson et al., 2012), in part through the regulation of Wnt5a
expression, a gene that when inactivated in mouse affects external ear
development (Qian et al., 2007). Altogether, these data uncover roles
for BMP and Wnt signalling in instructing external ear morphogenesis
downstream of Hoxa2; how these pathways interact remains to be
determined. More generally, understanding how multiple genes
integrate into functional networks is becoming key to a full
comprehension of the molecular processes that underlie normal and
defective external ear morphogenesis.
Acknowledgements
We thank F. Santagati and A. Vitobello for experimental assistance and
discussion; B. Hogan for the kind gift of the Bmp4 conditional allele; and L.
Fasano for the Tshz2 probe.
Funding
M.M. was supported by the Faculté de Chirurgie Dentaire de Strasbourg. S.A.
was supported by a grant of the Biotechnology and Biological Sciences
Development
Hoxa2 and outer ear development
Research Council [BB/H018123/2] to N.B. Work in the F.M.R. laboratory is
supported by the Swiss National Science Foundation [Sinergia
CRSI33_127440], Fondation pour l’Aide à la Recherche sur la Sclérose en
Plaques (ARSEP) and the Novartis Research Foundation.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
M.M. carried out most of the experiments. C.F.K. performed the 3D
reconstruction of the external ear. S.D. generated the Rosa(lox-stop-lox)Hoxa2-IRES-EGFP
allele. S.A. carried out ChIP/qPCR assays. N.V. performed some in situ
hybridisation experiments. T.K., H.K. and N.B. contributed to data analysis and
discussion as well as sharing unpublished results. F.M.R. and M.M. designed
the experiments, analysed the data and wrote the manuscript.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.098046/-/DC1
References
Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C.,
Weil, D., Cruaud, C., Sahly, I., Leibovici, M. et al. (1997). A human
homologue of the Drosophila eyes absent gene underlies branchio-oto-renal
(BOR) syndrome and identifies a novel gene family. Nat. Genet. 15, 157-164.
Alasti, F. and Van Camp, G. (2009). Genetics of microtia and associated
syndromes. J. Med. Genet. 46, 361-369.
Alasti, F., Sadeghi, A., Sanati, M. H., Farhadi, M., Stollar, E., Somers, T. and
Van Camp, G. (2008). A mutation in HOXA2 is responsible for autosomalrecessive microtia in an Iranian family. Am. J. Hum. Genet. 82, 982-991.
Baschek, V., Steinert, W. and Hildebrant, L. (2006). External ear duplication, a
rare branchial arch abnormality. Laryngorhinootologie 85, 861.
Caubit, X., Coré, N., Boned, A., Kerridge, S., Djabali, M. and Fasano, L. (2000).
Vertebrate orthologues of the Drosophila region-specific patterning gene
teashirt. Mech. Dev. 91, 445-448.
Couly, G., Grapin-Botton, A., Coltey, P., Ruhin, B. and Le Douarin, N. M.
(1998). Determination of the identity of the derivatives of the cephalic neural
crest: incompatibility between Hox gene expression and lower jaw
development. Development 125, 3445-3459.
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A.
P. (1998). Modification of gene activity in mouse embryos in utero by a
tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8, 1323-1326.
Donaldson, I. J., Amin, S., Hensman, J. J., Kutejova, E., Rattray, M.,
Lawrence, N., Hayes, A., Ward, C. M. and Bobola, N. (2012). Genome-wide
occupancy links Hoxa2 to Wnt-β-catenin signaling in mouse embryonic
development. Nucleic Acids Res. 40, 3990-4001.
Dupé, V., Davenne, M., Brocard, J., Dollé, P., Mark, M., Dierich, A., Chambon,
P. and Rijli, F. M. (1997). In vivo functional analysis of the Hoxa-1 3⬘ retinoic
acid response element (3’RARE). Development 124, 399-410.
Gendron-Maguire, M., Mallo, M., Zhang, M. and Gridley, T. (1993). Hoxa-2
mutant mice exhibit homeotic transformation of skeletal elements derived
from cranial neural crest. Cell 75, 1317-1331.
Gore, S. M., Myers, S. R. and Gault, D. (2006). Mirror ear: a reconstructive
technique for substantial tragal anomalies or polyotia. J. Plast. Reconstr. Aesthet.
Surg. 59, 499-504.
Grammatopoulos, G. A., Bell, E., Toole, L., Lumsden, A. and Tucker, A. S.
(2000). Homeotic transformation of branchial arch identity after Hoxa2
overexpression. Development 127, 5355-5365.
Heldin, C. H. and Moustakas, A. (2012). Role of Smads in TGFβ signaling. Cell
Tissue Res. 347, 21-36.
Hogan, B. L., Blessing, M., Winnier, G. E., Suzuki, N. and Jones, C. M. (1994).
Growth factors in development: the role of TGF-beta related polypeptide
signalling molecules in embryogenesis. Dev. Suppl. 1994, 53-60.
Horbelt, D., Denkis, A. and Knaus, P. (2012). A portrait of Transforming Growth
Factor β superfamily signalling: background matters. Int. J. Biochem. Cell Biol.
44, 469-474.
Hunter, A. G. and Yotsuyanagi, T. (2005). The external ear: more attention to
detail may aid syndrome diagnosis and contribute answers to embryological
questions. Am. J. Med. Genet. 135A, 237-250.
Jakubíková, J., Staník, R. and Staníková, A. (2005). Malformations of the first
branchial cleft: duplication of the external auditory canal. Int. J. Pediatr.
Otorhinolaryngol. 69, 255-261.
Kagurasho, M., Yamada, S., Uwabe, C., Kose, K. and Takakuwa, T. (2012).
Movement of the external ear in human embryo. Head Face Med. 8, 2.
King, J. A., Marker, P. C., Seung, K. J. and Kingsley, D. M. (1994). BMP5 and the
molecular, skeletal, and soft-tissue alterations in short ear mice. Dev. Biol. 166,
112-122.
Development 140 (21)
Kingsley, D. M., Bland, A. E., Grubber, J. M., Marker, P. C., Russell, L. B.,
Copeland, N. G. and Jenkins, N. A. (1992). The mouse short ear skeletal
morphogenesis locus is associated with defects in a bone morphogenetic
member of the TGF beta superfamily. Cell 71, 399-410.
Klockars, T. and Rautio, J. (2009). Embryology and epidemiology of microtia.
Facial Plast. Surg. 25, 145-148.
Kochhar, A., Fischer, S. M., Kimberling, W. J. and Smith, R. J. (2007). Branchiooto-renal syndrome. Am. J. Med. Genet. A 143A, 1671-1678.
Köntges, G. and Lumsden, A. (1996). Rhombencephalic neural crest
segmentation is preserved throughout craniofacial ontogeny. Development
122, 3229-3242.
Kountakis, S. E., Helidonis, E. and Jahrsdoerfer, R. A. (1995). Microtia grade as
an indicator of middle ear development in aural atresia. Arch. Otolaryngol.
Head Neck Surg. 121, 885-886.
Ku, P. K., Tong, M. C. and Yue, V. (1998). Polyotia – a rare external ear anomaly.
Int. J. Pediatr. Otorhinolaryngol. 46, 117-120.
Kulessa, H. and Hogan, B. L. (2002). Generation of a loxP flanked bmp4loxPlacZ allele marked by conditional lacZ expression. Genesis 32, 66-68.
Kuratani, S. (2005). Craniofacial development and the evolution of the
vertebrates: the old problems on a new background. Zoolog. Sci. 22, 1-19.
Lobe, C. G., Koop, K. E., Kreppner, W., Lomeli, H., Gertsenstein, M. and
Nagy, A. (1999). Z/AP, a double reporter for cre-mediated recombination. Dev.
Biol. 208, 281-292.
Madisen, L., Zwingman, T. A., Sunkin, S. M., Oh, S. W., Zariwala, H. A., Gu, H.,
Ng, L. L., Palmiter, R. D., Hawrylycz, M. J., Jones, A. R. et al. (2010). A robust
and high-throughput Cre reporting and characterization system for the whole
mouse brain. Nat. Neurosci. 13, 133-140.
Mallo, M. and Gridley, T. (1996). Development of the mammalian ear:
coordinate regulation of formation of the tympanic ring and the external
acoustic meatus. Development 122, 173-179.
Mark, M., Lohnes, D., Mendelsohn, C., Dupé, V., Vonesch, J. L., Kastner, P.,
Rijli, F., Bloch-Zupan, A. and Chambon, P. (1995). Roles of retinoic acid
receptors and of Hox genes in the patterning of the teeth and of the jaw
skeleton. Int. J. Dev. Biol. 39, 111-121.
Martin, J. F., Bradley, A. and Olson, E. N. (1995). The paired-like homeo box
gene MHox is required for early events of skeletogenesis in multiple lineages.
Genes Dev. 9, 1237-1249.
Meyer, L. R., Zweig, A. S., Hinrichs, A. S., Karolchik, D., Kuhn, R. M., Wong,
M., Sloan, C. A., Rosenbloom, K. R., Roe, G., Rhead, B. et al. (2013). The
UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids
Res. 41, D64-D69.
Miguez, A., Ducret, S., Di Meglio, T., Parras, C., Hmidan, H., Haton, C.,
Sekizar, S., Mannioui, A., Vidal, M., Kerever, A. et al. (2012). Opposing roles
for Hoxa2 and Hoxb2 in hindbrain oligodendrocyte patterning. J. Neurosci. 32,
17172-17185.
Minoux, M. and Rijli, F. M. (2010). Molecular mechanisms of cranial neural crest
cell migration and patterning in craniofacial development. Development 137,
2605-2621.
Minoux, M., Antonarakis, G. S., Kmita, M., Duboule, D. and Rijli, F. M. (2009).
Rostral and caudal pharyngeal arches share a common neural crest ground
pattern. Development 136, 637-645.
Mishra, S. C. and Misra, M. (1978). Mirror image pinna. J. Laryngol. Otol. 92, 709711.
Oury, F., Murakami, Y., Renaud, J. S., Pasqualetti, M., Charnay, P., Ren, S. Y.
and Rijli, F. M. (2006). Hoxa2- and rhombomere-dependent development of
the mouse facial somatosensory map. Science 313, 1408-1413.
Pan, B., Qie, S., Zhao, Y., Tang, X., Lin, L., Yang, Q., Zhuang, H. and Jiang, H.
(2010). Surgical management of polyotia. J. Plast. Reconstr. Aesthet. Surg. 63,
1283-1288.
Pasqualetti, M., Ori, M., Nardi, I. and Rijli, F. M. (2000). Ectopic Hoxa2
induction after neural crest migration results in homeosis of jaw elements in
Xenopus. Development 127, 5367-5378.
Pasqualetti, M., Ren, S. Y., Poulet, M., LeMeur, M., Dierich, A. and Rijli, F. M.
(2002). A Hoxa2 knockin allele that expresses EGFP upon conditional Cremediated recombination. Genesis 32, 109-111.
Passos-Bueno, M. R., Ornelas, C. C. and Fanganiello, R. D. (2009). Syndromes
of the first and second pharyngeal arches: A review. Am. J. Med. Genet. A 149A,
1853-1859.
Porter, C. J. and Tan, S. T. (2005). Congenital auricular anomalies: topographic
anatomy, embryology, classification, and treatment strategies. Plast. Reconstr.
Surg. 115, 1701-1712.
Qian, D., Jones, C., Rzadzinska, A., Mark, S., Zhang, X., Steel, K. P., Dai, X.
and Chen, P. (2007). Wnt5a functions in planar cell polarity regulation in mice.
Dev. Biol. 306, 121-133.
Ren, S. Y., Pasqualetti, M., Dierich, A., Le Meur, M. and Rijli, F. M. (2002). A
Hoxa2 mutant conditional allele generated by Flp- and Cre-mediated
recombination. Genesis 32, 105-108.
Rijli, F. M., Mark, M., Lakkaraju, S., Dierich, A., Dollé, P. and Chambon, P.
(1993). A homeotic transformation is generated in the rostral branchial region
Development
4396 RESEARCH ARTICLE
of the head by disruption of Hoxa-2, which acts as a selector gene. Cell 75,
1333-1349.
Santagati, F. and Rijli, F. M. (2003). Cranial neural crest and the building of the
vertebrate head. Nat. Rev. Neurosci. 4, 806-818.
Santagati, F., Minoux, M., Ren, S. Y. and Rijli, F. M. (2005). Temporal
requirement of Hoxa2 in cranial neural crest skeletal morphogenesis.
Development 132, 4927-4936.
Schoenwolf, G. C. and Larsen, W. J. (2009). Larsen’s Human Embryology, 4th edn.
Philadelphia, PA: Elsevier/Churchill Livingstone.
Solloway, M. J. and Robertson, E. J. (1999). Early embryonic lethality in
Bmp5;Bmp7 double mutant mice suggests functional redundancy within the
60A subgroup. Development 126, 1753-1768.
Takechi, M. and Kuratani, S. (2010). History of studies on mammalian middle
ear evolution: a comparative morphological and developmental biology
perspective. J. Exp. Zool. 314B, 417-433.
RESEARCH ARTICLE 4397
ten Berge, D., Brouwer, A., Korving, J., Martin, J. F. and Meijlink, F. (1998).
Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and
limbs. Development 125, 3831-3842.
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone
morphogenetic protein-4 is required for mesoderm formation and patterning
in the mouse. Genes Dev. 9, 2105-2116.
Wood-Jones, F. and I-Chuan, W. (1934). The development of the external ear. J.
Anat. 68, 525-533.
Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S. and Maas, R. (1999).
Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of
organ primordia. Nat. Genet. 23, 113-117.
Zakin, L. and De Robertis, E. M. (2004). Inactivation of mouse Twisted
gastrulation reveals its role in promoting Bmp4 activity during forebrain
development. Development 131, 413-424.
Development
Hoxa2 and outer ear development
Fig. S1. Fate mapping of neural crest-derived pinna. (A-L) Anti-EGFP (A, B, D) and anti-RFP (E, F, H, I, J, L)
immunostaining and DAPI staining (A, C, D, E, G, H, I, K, L) on frontal (A-D, E-H) and horizontal (I-L) sections through the
external ear of E14.5 Wnt1::Cre;Hoxa2EGFP(lox-neo-lox)/+ (A-D) and R4::Cre;Rosa-CAG-LSL-tdTomato (E-L) fetuses. In A and
D, E and H, I and L, DAPI and anti-EGFP (A, D) or DAPI and anti-RFP (E, H, I, L) stainings are merged. B-D, F-H and J-L
are higher magnifications of the domains outlined in A, E and I, respectively.
Fig. S2. Temporal requirement of Hoxa2 in pinna morphogenesis. (A-D) Lateral views of the head of E18.5 wild-type
(WT) (A, C) and CMV::CreERT2;Hoxa2lox/del mutant fetuses treated with tamoxifen (TM) at E12.5, E13.0 and E13.5 (B, D).
C and D are enlarged views of pinnae (arrows, Pi) in A and B, respectively. Note the small pinna resulting from late Hoxa2
downregulation (B, D).
Fig. S3. Loss of Hoxa2 expression in TM-treated mutants. (A-F) Hoxa2 in situ hybridization on frontal sections
through the external ear of E14.5 Hoxa2lox/+ control (A-C) and CMV::CreERT2;Hoxa2lox/del mutant (D-F) fetuses treated with
tamoxifen (TM) at E12.5, E13.0 and E13.5. Top is dorsal, bottom is ventral and sections are from anterior to posterior.
Note the significant downregulation of Hoxa2 expression in D-F.
Fig. S4. Distribution of phosphoSmad1/5/8, Bmp4 and Bmp5 expression in the developing pinna. (A-C) Bmp4
(A) and Bmp5 (B) in situ hybridization and anti-phosphoSmad1/5/8 antibody immunostaining (C) on adjacent horizontal
sections through the external ear of E14.5 wild-type (WT) fetuses. Black arrows in A and B, and the white arrow in C
show Bmp4, Bmp5 and phosphoSmad1/5/8 positive cells at the base the pinna. The white arrowhead in C shows the
Eya1+ subpopulation of phosphoSmad1/5/8 positive cells next to the pinna mesenchymal core of Bmp5 expression. The
green arrowhead in C shows phosphoSmad1/5/8 activated cells at the tip of the pinna, just beneath the ectoderm. Top is
anterior, bottom is posterior.
Fig. S5. Hoxa2 positively regulates Tsg. (A-O) In situ hybridization on adjacent horizontal sections through the external
ear of E14.5 wild-type (WT) fetuses using Bmp4 (A, D, G, J, M) and Tsg (B, E, H, K, N) probes. A merge between adjacent
sections hybridised with Bmp4 and Tsg, respectively (C, F, I, L, O) reveals that Tsg is highly expressed directly adjacent
to the Bmp4 positive domain. (P-R) Tsg in situ hybridization on horizontal sections through the external ear of E14.5
CMV::CreERT2;Hoxa2lox/del mutant fetuses treated with tamoxifen (TM) at E12.5, E13.0 and E13.5. Top is anterior, bottom is
posterior.
Fig. S6. Validation by qPCR of Hoxa2 binding at Bmp4 and Bmp5 non-coding regions. (A) Fold enrichment of Hoxa2
over IgG negative control antibody (Neg Ab) is shown for each Hoxa2-bound region. Values correspond to the average
of duplicate samples and are representative of two independent experiments. Itih4 is a negative control gene (unbound
region). The numbers in brackets correspond to the FDR (False Discovery Rate) of each bound region in Hoxa2 ChIP-seq
(Donaldson et al., 2012). (B) Sequence alignment and conservation of Hoxa2-bound regions across Vertebrates, provided
by the UCSC browser (Meyer et al., 2013).
Fig. S7. Tsg is ectopically expressed in the Hoxa2-induced duplicated pinna. (A, B) Tsg in situ hybridization
performed on horizontal sections through the external ear of E14.5 Wild type (WT) (A) and Wnt1Hoxa2-IRES-EGFP mutant (B)
fetuses. Top is anterior, bottom is posterior. The black arrows indicate Tsg ectopic expression in the duplicated pinna (Pi*)
and more anteriorly.